阅读说明：本技术 一种快速分离免疫磁珠的方法 (method for quickly separating immunomagnetic beads ) 是由 姜宇腾 于 2019-08-13 设计创作，主要内容包括：本发明公开了快速分离免疫磁珠的方法,可应用于化学发光仪和核酸提取仪。是将包被抗体/抗原的免疫磁珠和标记物标记的抗体/抗原以及待测物在反应杯中孵育；反应结束后,加入小型永小型永磁体吸附免疫磁珠,使抗原-抗体-磁珠免疫复合物从反应液中分离；清洗液清洗后加入发光底物/激发系统促使反应杯内发光物质释放光子,进行光子计数检测发光强度。本发明将小型永磁体置入反应液内,使永小型永磁体和反应液中的免疫磁珠直接接触,在极短时间(3-15s)内实现快速分离。分离速度快,效率高,免疫磁珠流失率大幅度降低,并且大大简化了仪器设计过程中磁分离模块的结构,缩短了免疫磁珠从反应液中分离和清洗的时间。(the invention discloses a method for quickly separating immunomagnetic beads, which can be applied to a chemiluminescence apparatus and a nucleic acid extraction apparatus. Incubating immune magnetic beads coated with antibody/antigen, antibody/antigen marked by a marker and a substance to be detected in a reaction cup; after the reaction is finished, adding a small permanent magnet to adsorb the immunomagnetic beads, and separating the antigen-antibody-magnetic bead immune compound from the reaction solution; after cleaning, adding a luminescent substrate/excitation system to promote the luminescent substance in the reaction cup to release photons, and carrying out photon counting to detect the luminous intensity. The invention puts the small permanent magnet into the reaction liquid, so that the permanent small permanent magnet is directly contacted with the immunomagnetic beads in the reaction liquid, and the rapid separation is realized in a very short time (3-15 s). The separation speed is fast, the efficiency is high, the loss rate of the immunomagnetic beads is greatly reduced, the structure of a magnetic separation module in the design process of an instrument is greatly simplified, and the time for separating and cleaning the immunomagnetic beads from reaction liquid is shortened.)

1. a method for rapidly separating immunomagnetic beads is characterized by comprising the following steps:

(1) Placing the immunomagnetic beads, the antigen/antibody marked by the marker and the substance to be detected in a reaction cup, and incubating to form an antigen-antibody-magnetic bead immune complex;

(2) After the incubation is finished, adding a small permanent magnet into the reaction liquid in the reaction cup, and adsorbing the immunomagnetic bead antigen-antibody-magnetic bead immune complex combined with the marked antigen/antibody and the object to be detected onto the surface of the small permanent magnet within 3-15 seconds to realize the process from dispersion to aggregation;

(3) and cleaning with a cleaning solution, adding a luminescent substrate solution or an excitant solution to promote the luminescent substance in the reaction cup to release photons, carrying out photon counting to detect the luminous intensity, and calculating the content of the substance to be detected through a standard curve.

2. A method for rapid separation of immunomagnetic beads according to claim 1, wherein the label in step (1) is a luminescent substance such as acridinium esters, luminol, oxalyl peroxide, ruthenium terpyridyl, enzymes such as alkaline phosphatase and horseradish peroxidase.

3. the method for rapidly separating immunomagnetic beads according to claim 1, wherein the small permanent magnet in step (2) has a diameter of 0.05-10mm, is a bare small permanent magnet or a small permanent magnet with a surface protective layer, and can be used alone or in combination.

4. the method for rapidly separating immunomagnetic beads according to claim 1, wherein the washing solution in step (3) is weak acid, weak base or neutral buffer containing surfactant.

5. The method of claim 1, wherein the luminescent substrate solution in step (3) is AMPPD-enhancer solution or CSPD-enhancer solution, luminol-H₂O₂-an enhancer solution.

6. the method of claim 1, wherein the trigger solution in step (3) is nitric acid-H added sequentially₂O₂mixing the solution, and adding a NaOH solution of a surfactant or a dibutylethanolamine solution.

Technical Field

The invention relates to a method for quickly separating immunomagnetic beads, which changes a small permanent magnet from the outside of a reaction cup into the inside of the reaction cup, and belongs to the field of immunological detection.

Background

The immunological detection method is a series of experimental methods for measuring antigen, antibody, immune cell and cell factor secreted by the immune cell, which are designed by applying the immunological theory. Along with the mutual infiltration among disciplines, the related range of immunology is continuously expanded, and new immunology detection methods are in the endlessly. The application range of immunological methods is also expanding day by day, which not only becomes an important method for diagnosing various clinical diseases, but also provides convenience for the research of numerous disciplines.

Chemiluminescence is one of the most important subdivisions in the in vitro diagnosis industry, the application of chemiluminescence in the aspect of immunological detection is increasingly emphasized by people, the method has the advantages of high sensitivity, wide linearity, quick reaction, few influencing factors, accurate result and the like, and various chemiluminescent markers are as follows: direct chemiluminescence, the marker being acridinium ester (yapei) or ABEI (new industry); enzymatic chemiluminescence, and the marker is alkaline phosphatase (mansion wave) or horseradish peroxidase (vigorous); electrochemiluminescence, and the marker is terpyridyl ruthenium (Roche).

The chemiluminescence technology based on magnetic particles is a more advanced detection technology in immunodiagnosis and is also the mainstream of chemiluminescence immunoassay at present. The immunomagnetic microspheres can simply and rapidly enrich and remove cancer cells from blood or bone marrow, are widely applied to disease detection, cancer treatment and autologous bone marrow transplantation, and are also used for separating fetal cells from maternal peripheral blood for noninvasive prenatal diagnosis. Immunomagnetic bead separation technology used in microbial detection and capable of accurately and rapidly detecting in sampleColi O 157this is of great significance for food hygiene and prevention of disease transmission. The magnetic beads contain artificially synthesized magnetic cores inside and can be attracted by the magnetic force of the magnets; the external grafted functional group can be combined with active protein (antibody) to be used as a solid phase carrier. When the antibody on the magnetic bead is combined with corresponding microbe or specific antigen matter, antigen-antibody-magnetic bead immune complex is formed, which has high magnetic responsiveness and moves directionally under the action of magnetic force to separate the complex from other matter, so as to separate, concentrate and purify microbe or specific antigen matter. FIG. 1 is a schematic view of a conventional magnetic separation apparatus; the magnetic separation device is a mechanical arm for fixing a small permanent magnet, and is far away from the reaction cup when the reaction solution in the reaction cup is incubated; after the incubation is finished, the mechanical arm moves the small permanent magnet to the side surface of the reaction cup, and the antigen-antibody-magnetic bead immune complex is separated from the reaction liquid. FIG. 5 is a robotic arm-magnet apparatus of publication No. CN 105758848A entitled incubated magnetic separation apparatus for use in chemiluminescent assays; FIG. 6 is a robot arm-magnet apparatus for use in a luminescent immunomagnetic separation cleaning device, publication No. CN 208019096U. In short, the permanent magnet or the electromagnet operates outside the light emitting tube or the microplate. It is known to magnetically divide the same kind of magnetic particlesthe stronger the magnetic field intensity, the faster the separation and the shorter the time. According to the following formula of the strength of the electromagnetic field,

The magnetic field strength is proportional to the magnet magnetism and inversely proportional to the magnetic circuit length. Because the magnet is contacted with the side surface of the reaction cup, the shortest distance between the magnet and the magnetic particles in the reaction liquid is separated from the wall of the reaction cup, the longest distance is the thickness and the inner diameter of the wall of the reaction cup, the average magnetic path length is longer, the integral magnetic field intensity of the reaction cup is weaker, and the magnetic separation time is about 3 min. The magnetizing quantity of the powerful permanent magnet used at present basically reaches the upper limit, and if the magnetic force is continuously increased, the technical difficulty and the cost are both greatly increased (namely, the N x I in the formula basically tends to a fixed value). Therefore, under the condition that the magnetic force is not changed, the length of the magnetic circuit is effectively shortened, and the magnetic separation device is an effective means for realizing rapid magnetic separation. After magnetic separation, the reaction solution was removed, and washing was repeated 5 times with a washing solution. Because the antigen-antibody-magnetic bead immune complex is not firmly adsorbed by the magnet, a part of the complex can be dispersed in the process of adding the cleaning solution every time, the loss of the antigen-antibody-magnetic bead immune complex is avoided, and the magnetic separation is continued for more than 1min before removing the cleaning solution, so that the whole detection process (reaction incubation, liquid injection, magnetic separation and cleaning solution) is used for at least more than 10 min. This, of course, does not affect the conventional chemiluminescence detection mode at regular test speeds (20-40 minutes) much, but severely affects the test speed at fast chemiluminescence (3-7 min). Therefore, to obtain the detection result as soon as possible, shortening the magnetic separation time and the washing time of the antigen-antibody-magnetic bead immunocomplex is a key factor.

Disclosure of Invention

The magnetic separation method adopted by the prior chemiluminescence instrument mainly arranges a permanent magnet/an electromagnet outside a reaction cup, belongs to non-contact magnetic separation, and has longer magnetic separation time and poorer separation effect of magnetic particles due to the relatively larger distance between the magnet and reaction liquid and the thickness of the wall of the reaction cup. In order to overcome the defects of the prior chemiluminescence apparatus, the invention provides a method for quickly separating immunomagnetic beads, which is characterized in that a magnet device is arranged outside a reaction cup, a small permanent magnet is arranged inside the reaction cup, the separation speed is high, the efficiency is high, the time for magnetically separating and cleaning reaction liquid is shortened, the clinical quick judgment is facilitated, and the structure of a magnetic separation module in the design process of the apparatus is simplified.

In order to achieve the purpose of the invention, the following technical scheme is adopted in the application:

A method for rapidly separating immunomagnetic beads comprises the following steps:

(1) adding immunomagnetic beads, labeled antigen/antibody and a substance to be detected into the reaction cup, and incubating to obtain an antigen-antibody-magnetic bead immune complex;

The reaction cup can be a single or a light-emitting tube and an enzyme label plate which are connected together, and other similar reaction containers;

The small permanent magnet is selected according to the size of the reaction cup, and the diameter of the small permanent magnet is 0.05-10 mm;

The small permanent magnet is an exposed small permanent magnet, and can also be a small permanent magnet with a protective layer coated on the surface;

The protective layer can be a polymer coating layer such as polytetrafluoroethylene, polystyrene, polymethyl methacrylate, polyethylene, polypropylene, polyvinyl chloride, nylon plastic, ABS plastic and the like, a coating layer such as Teflon, ceramic, paint and the like, a coating layer such as copper, silver, gold, platinum and the like, and other similar protective layer substances.

the small permanent magnet is not limited in shape and can be spherical, rugby-ball, cylindrical, conical, star-shaped, elongated, cluster-shaped, regular and irregular polyhedrons, and the like.

The magnet is not limited in kind, and can be a permanent magnet, a small samarium-cobalt permanent magnet, a neodymium-iron-boron magnet (strong magnet),Ferrite magnetalnico magnet, ferrochrome-cobalt magnet.

The antigen/antibody can be complete or partial antigen/antibody which is of animal source, human source and expressed by gene recombination;

The label for labeling the antibody may be one of biotin, an isotope, an enzyme, a luminescent substance, and a fluorescent substance;

For example: the labelled isotope is³H、¹⁸O、⁴²K、¹³¹I、³⁵S、³²P、¹⁵n, and the like.

For example: the labeling fluorescent substance is one or more of FITC, rare earth element complex, CY series and Alexa series fluorescent protein dyes.

For example: the labeled biological enzyme is Horse Radish Peroxidase (HRP), Alkaline Phosphatase (AP) and the like;

(2) after the reaction is finished, adding a permanent small permanent magnet into the reaction cup, and aggregating the combined sample originally dispersed in the reaction liquid and the immunomagnetic beads (antigen-antibody-magnetic bead immunocomplexes) for marking the antigen/antibody in the reaction liquid;

(3) Cleaning with cleaning solution, separating, adding luminescent substrate solution or excitant solution to make luminescent substance in the reaction cup release photon, measuring photon quantity with chemiluminescence apparatus, recording luminescence value, obtaining standard curve, and obtaining sample content according to the standard curve.

the luminescent substrate solution is AMPPD-enhancer or CSPD-enhancer (alkaline phosphatase), luminol-H2O 2-enhancer solution (horseradish peroxidase);

the excimer solution can be nitric acid-H₂O₂NaOH-surfactant, dibutylethanolamine solution;

the small permanent magnet is placed in the reaction liquid in the reaction cup after full reaction, the small permanent magnet is directly contacted with the reaction liquid, the magnetic particle small permanent magnet is combined under the action of magnetic force, the maximum distance between the small permanent magnet and the reaction liquid is half of the inner diameter of the reaction cup, the average magnetic path length is small, the integral magnetic field in the reaction cup is very strong, and the time for complete magnetic separation is only 3-10 s. Because the small permanent magnet is firmly combined with the antigen-antibody-magnetic bead immune complex, the complex can not be dispersed when washing liquor is added or discarded, the washing liquor is not needed to be separated in a magnetic way after the washing liquor is added, the washing liquor can be immediately removed, the washing time is greatly shortened, and the whole detection process (incubation, magnetic separation and washing) is used within 3-5 min.

The direct contact type rapid magnetic separation method is suitable for instrument platforms such as a tubular chemiluminescence instrument, a plate-type chemiluminescence instrument and a nucleic acid extraction instrument. The traditional external magnetic-ferromagnetic separation method has higher requirements on a magnetic separation module of an instrument, not only needs to arrange a mechanical arm to control the running track of a magnet, but also needs a matched software program, and has a complex structure and higher cost. The built-in magnetic-magnetic separation method can realize separation only by adding one or more small permanent magnets into reaction liquid after the incubation reaction of the reaction cup is finished, thereby greatly simplifying the structure of the instrument, simplifying and miniaturizing the chemiluminescence apparatus, reducing the fault of the instrument, improving the reliability and accuracy of test results, greatly shortening the detection time, making the instantaneization of the experimental report result possible, and having great significance for quick diagnosis and treatment of patients by clinical doctors in hospitals.

in summary, compared with the prior art, the invention has the following advantages:

(1) The invention changes the arrangement mode of the magnet when the immunomagnetic beads are separated, the magnet is changed from the outside of the reaction cup to the inside of the reaction cup, and the non-contact magnetic separation mode is changed to the direct contact magnetic separation mode; the invention directly puts the small permanent magnet into the luminotron or the reaction cup, because the distance between each position of the reaction liquid and the magnet is obviously shortened, the time for completely separating the immunomagnetic beads from the reaction liquid is greatly shortened, the time can reach within 15s at the fastest, the magnetic separation and cleaning time of the experiment is shortened, and the cleaning liquid is cleaned and discarded, thereby avoiding the process of re-gathering after the magnetic particles are scattered, being capable of obtaining the diagnosis result more quickly, greatly contributing to the quick clinical judgment, and leading the disease to be diagnosed as early as possible and treated better.

(2) the small permanent magnet is directly placed in the reaction cup, so that a magnetic separation component of a chemiluminescence instrument can be omitted, hardware and software are simplified, the fault rate of the instrument is reduced, the accuracy and reliability of detection are improved, the miniaturization and automation of the instrument are facilitated, and the instrument cost is reduced.

(3) The chemiluminescence apparatus and the matched diagnostic kit thereof prepared by the invention can be used for detecting the result of the first sample in 4-6 minutes at the fastest speed in clinical examination, greatly improve the testing speed and the sample flux, realize the rapid acquisition of the diagnostic result at the side of a patient, and enable the disease to be diagnosed as early as possible and treated better.

(4) The invention is suitable for experimental methods such as a double-antibody sandwich method, a double-antigen sandwich method, a competition method, an indirect method and the like, and can be used for nucleic acid extraction, environmental monitoring, biological analysis, drug screening and the like.

drawings

FIG. 1 is a schematic view of a conventional magnetic separation apparatus; wherein: 1-luminotron, 2-magnet;

FIG. 2 is a top view of FIG. 1;

FIG. 3 is a schematic view of the magnetic separation apparatus of the present invention;

FIG. 4 is a top view of FIG. 3; 3-permanent magnetic beads;

FIG. 5 is a schematic view of a conventional magnetic separation apparatus disclosed in publication No. CN 105758848A, and the inside of the box is divided into a robot-magnet apparatus;

FIG. 6 is a schematic view of a conventional magnetic separation device disclosed in CN 208019096U, and a mechanical arm-magnet device is arranged in a box;

FIG. 7 shows the results of chemiluminescence assay in example 1 using the acridinium ester double-antibody sandwich method;

FIG. 8 shows the results of the alkaline phosphatase competition method chemiluminescence assay in example 2.

Detailed Description

The following examples are presented to assist those skilled in the art in a more complete understanding of the present invention and are not intended to limit the invention in any way.

the small permanent magnet adopted by the invention can be customized according to the required size and the required magnetic force, the magnet components are consistent with the magnetic components of magnetic particles (namely immunomagnetic beads) in the kit, the small permanent magnet is cleaned and disinfected before use without antigen, the pollution problem caused by the built-in small permanent magnet is not worried, and after detection, repeated experiments are not required to be recovered, and the small permanent magnet is abandoned together with an ELISA plate or a light-emitting tube which is used as an easily-consumed product of experimental equipment.